Purification of vaccinia virus- and recombinant vaccinia virus-based vaccines
The present invention relates to methods for purification of Vaccinia viruses (VV) and/or Vaccinia virus (VV) particles, which can lead to highly pure and stable virus preparations of predominantly biologically active viruses. The invention encompasses purifying a virus preparation in a sterilized way with high efficiency and desirable yield in terms of purity, biological activity and stability, aspects advantageous for industrial production.
1. A method for the purification of biologically active Vaccinia virus comprising:
i) loading a solid-phase matrix, to which a ligand is attached, with a biologically active Vaccinia virus contained in a liquid-phase culture,
wherein the matrix with attached ligand is a sulfated cellulose;
ii) washing the matrix; and
iii) eluting the biologically active Vaccinia virus,
wherein the method reduces the amount of dsDNA in the eluted virus to less than 5% of input.
2. The method of claim 1 , wherein the method is an industrial-scale process.
3. The method of claim 1 , wherein the method is aseptic.
4. The method of claim 1 , wherein the Vaccinia virus is a recombinant Vaccinia virus.
5. The method of claim 1 , wherein the Vaccinia virus is MVA or recombinant MVA.
6. The method of claim 1 , wherein the solid-phase matrix comprises or is a membrane.
7. The method of claim 6 , wherein the solid-phase matrix comprises or is a sulfated reinforced cellulose membrane.
8. The method of claim 1 , wherein the matrix comprises a pore size of greater than 0.25 μm.
9. The method of claim 1 , wherein contaminants are removed from the Vaccinia virus in the liquid-phase culture.
10. The method of claim 1 , wherein the Vaccinia virus is eluted with sodium chloride (NaCl).
11. The method of claim 10 , wherein the Vaccinia virus is eluted by an increasing NaCl concentration gradient ranging from 0.15 M to 2.0 M.
12. The method of claim 10 , wherein the Vaccinia virus is eluted with 2.0 M NaCl.
13. The method of claim 1 , additionally comprising a purification step by ion-exchange.
14. The method of claim 13 , wherein the purification step by ion-exchange comprises a membrane.
15. The method of claim 14 , wherein the method reduces the amount of dsDNA in the eluted virus to less than 0.1% of input.
16. The method of claim 1 , wherein the pH value of the virus preparation is adjusted to a pH ranging from 4.0-11.0.
17. The method of claim 1 , further comprising administering the eluted Vaccinia virus to an animal.
18. The method of claim 16 , wherein the animal is a human.